Novel Target Discovery and Validation in Renal Cell Carcinoma

Renal cell carcinoma (RCC) is the 7th most common cancer in the UK. Almost one third of patients will have locally advanced or metastatic disease at presentation and a similar proportion will relapse despite undergoing surgery with a curative intent. More effective treatments are now available, although resistance is typically observed within months of starting treatment and median survival remains in the order of two years, highlighting the need for continued progress. The aim of this work was the discovery and further investigation of novel therapeutic targets in ccRCC through proteomic approaches. Two potential emerging targets were further investigated in ccRCC. Spleen tyrosine kinase (SYK) was identified to be alternatively spliced in ccRCC compared to normal tissue. SYK inhibition with R406 caused cell death at concentrations >1 μM. Transient Receptor Potential Canonical (TRPC) channels 1, 4 and 5 expression was variable at an mRNA level in primary ccRCC and normal kidney tissue but the lack of antibodies to accurately detect the TRPC proteins using Western blot limited detection at a protein level. The activation and inhibition of these channels in-vitro was explored in the A498 RCC cell line. A comprehensive proteomic analysis of ccRCC tissue compared with normal kidney tissue, selected on the basis of underlying genomic changes was undertaken. Integration of the data led to the identification of a number of potential novel therapeutic targets. Cyclooxygenase-1 (COX-1) and proteasome subunit type beta-9 (PSMB9), were taken forward and their upregulation in ccRCC at a protein level were demonstrated. Inhibition of COX-q was shown to cause RCC cell line death. The genetics of RCC at a descriptive level is comprehensive, but has not yet led to advances in treatment options. This work has demonstrated a valid approach to the integration of genomic and proteomic data that may be adopted in future studies

Babies, bugs and brains : How the early microbiome associates with infant brain and behavior development

Growing evidence is demonstrating the connection between the microbiota gut-brain axis and neurodevelopment. Microbiota colonization occurs before the maturation of many neural systems and is linked to brain health. Because of this it has been hypothesized that the early microbiome interactions along the gut-brain axis evolved to promote advanced cognitive functions and behaviors. Here, we performed a pilot study with a multidisciplinary approach to test if the microbiota composition of infants is associated with measures of early cognitive development, in particular neural rhythm tracking; language (forward speech) versus non-language (backwards speech) discrimination; and social joint attention. Fecal samples were collected from 56 infants between four and six months of age and sequenced by shotgun metagenomic sequencing. Of these, 44 performed the behavioral Point and Gaze test to measure joint attention. Infants were tested on either language discrimination using functional near-infrared spectroscopy (fNIRS; 25 infants had usable data) or neural rhythm tracking using electroencephalogram (EEG; 15 had usable data). Infants who succeeded at the Point and Gaze test tended to have increased Actinobacteria and reduced Firmicutes at the phylum level; and an increase in Bifidobacterium and Eggerthella along with a reduction in Hungatella and Streptococcus at the genus level. Measurements of neural rhythm tracking associated negatively to the abundance of Bifidobacterium and positively to the abundance of Clostridium and Enterococcus for the bacterial abundances, and associated positively to metabolic pathways that can influence neurodevelopment, including branched chain amino acid biosynthesis and pentose phosphate pathways. No associations were found for the fNIRS language discrimination measurements. Although the tests were underpowered due to the small pilot sample sizes, potential associations were identified between the microbiome and measurements of early cognitive development that are worth exploring further.Arts, Faculty ofMedicine, Faculty ofOther UBCNon UBCBiochemistry and Molecular Biology, Department ofMicrobiology and Immunology, Department ofPsychology, Department ofReviewedFacultyResearcherGraduat

UK Multicenter Prospective Evaluation of the Leibovich Score in Localized Renal Cell Carcinoma: Performance has Altered Over Time.

OBJECTIVE: To examine changes in outcome by the Leibovich score using contemporary and historic cohorts of patients presenting with renal cell carcinoma (RCC) PATIENTS AND METHODS: Prospective observational multicenter cohort study, recruiting patients with suspected newly diagnosed RCC. A historical cohort of patients was examined for comparison. Metastasis-free survival (MFS) formed the primary outcome measure. Model discrimination and calibration were evaluated using Cox proportional hazard regression and the Kaplan-Meier method. Overall performance of the Leibovich model was assessed by estimating explained variation. RESULTS: Seven hundred and six patients were recruited between 2011 and 2014 and RCC confirmed in 608 (86%) patients. Application of the Leibovich score to patients with localized clear cell RCC in this contemporary cohort demonstrated good model discrimination (c-index = 0.77) but suboptimal calibration, with improved MFS for intermediate- and high-risk patients (5-year MFS 85% and 50%, respectively) compared to the original Leibovich cohort (74% and 31%) and a historic (1998-2006) UK cohort (76% and 37%). The proportion of variation in outcome explained by the model is low and has declined over time (28% historic vs 22% contemporary UK cohort). CONCLUSION: Prognostic models are widely employed in patients with localized RCC to guide surveillance intensity and clinical trial selection. However, the majority of the variation in outcome remains unexplained by the Leibovich model and, over time, MFS rates among intermediate- and high-risk classified patients have altered. These findings are likely to have implications for all such models used in this setting

Dysregulation at multiple points of the kynurenine pathway is a ubiquitous feature of renal cancer: implications for tumour immune evasion

Funder: EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: "Ideas" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013)); doi: https://doi.org/10.13039/100011199Funder: DH | National Institute for Health Research (NIHR); doi: https://doi.org/10.13039/501100000272Abstract: Background: Indoleamine 2,3-dioxygenase (IDO), the first step in the kynurenine pathway (KP), is upregulated in some cancers and represents an attractive therapeutic target given its role in tumour immune evasion. However, the recent failure of an IDO inhibitor in a late phase trial raises questions about this strategy. Methods: Matched renal cell carcinoma (RCC) and normal kidney tissues were subject to proteomic profiling. Tissue immunohistochemistry and gene expression data were used to validate findings. Phenotypic effects of loss/gain of expression were examined in vitro. Results: Quinolate phosphoribosyltransferase (QPRT), the final and rate-limiting enzyme in the KP, was identified as being downregulated in RCC. Loss of QPRT expression led to increased potential for anchorage-independent growth. Gene expression, mass spectrometry (clear cell and chromophobe RCC) and tissue immunohistochemistry (clear cell, papillary and chromophobe), confirmed loss or decreased expression of QPRT and showed downregulation of other KP enzymes, including kynurenine 3-monoxygenase (KMO) and 3-hydroxyanthranilate-3,4-dioxygenase (HAAO), with a concomitant maintenance or upregulation of nicotinamide phosphoribosyltransferase (NAMPT), the key enzyme in the NAD+ salvage pathway. Conclusions: Widespread dysregulation of the KP is common in RCC and is likely to contribute to tumour immune evasion, carrying implications for effective therapeutic targeting of this critical pathwa

Application of Genomic Sequencing to Refine Patient Stratification for Adjuvant Therapy in Renal Cell Carcinoma

International audiencePurpose: Patients with resected localized clear-cell renal cell carcinoma (ccRCC) remain at variable risk of recurrence. Incorporation of biomarkers may refine risk prediction and inform adjuvant treatment decisions. We explored the role of tumor genomics in this setting, leveraging the largest cohort to date of localized ccRCC tissues subjected to targeted gene sequencing. Experimental Design: The somatic mutation status of 12 genes was determined in 943 ccRCC cases from a multinational cohort of patients, and associations to outcomes were examined in a Discovery (n = 469) and Validation (n = 474) framework. Results: Tumors containing a von-Hippel Lindau (VHL) mutation alone were associated with significantly improved outcomes in comparison with tumors containing a VHL plus additional mutations. Within the Discovery cohort, those with VHL+0, VHL+1, VHL+2, and VHL+≥3 tumors had disease-free survival (DFS) rates of 90.8%, 80.1%, 68.2%, and 50.7% respectively, at 5 years. This trend was replicated in the Validation cohort. Notably, these genomically defined groups were independent of tumor mutational burden. Amongst patients eligible for adjuvant therapy, those with a VHL+0 tumor (29%) had a 5-year DFS rate of 79.3% and could, therefore, potentially be spared further treatment. Conversely, patients with VHL+2 and VHL+≥3 tumors (32%) had equivalent DFS rates of 45.6% and 35.3%, respectively, and should be prioritized for adjuvant therapy. Conclusions: Genomic characterization of ccRCC identified biologically distinct groups of patients with divergent relapse rates. These groups account for the ∼80% of cases with VHL mutations and could be used to personalize adjuvant treatment discussions with patients as well as inform future adjuvant trial design

POLR1A variants underlie phenotypic heterogeneity in craniofacial, neural, and cardiac anomalies

Heterozygous pathogenic variants in POLR1A, which encodes the largest subunit of RNA Polymerase I, were previously identified as the cause of acrofacial dysostosis, Cincinnati-type. The predominant phenotypes observed in the cohort of 3 individuals were craniofacial anomalies reminiscent of Treacher Collins syndrome. We subsequently identified 17 additional individuals with 12 unique heterozygous variants in POLR1A and observed numerous additional phenotypes including neurodevelopmental abnormalities and structural cardiac defects, in combination with highly prevalent craniofacial anomalies and variable limb defects. To understand the pathogenesis of this pleiotropy, we modeled an allelic series of POLR1A variants in vitro and in vivo. In vitro assessments demonstrate variable effects of individual pathogenic variants on ribosomal RNA synthesis and nucleolar morphology, which supports the possibility of variant-specific phenotypic effects in affected individuals. To further explore variant-specific effects in vivo, we used CRISPR-Cas9 gene editing to recapitulate two human variants in mice. Additionally, spatiotemporal requirements for Polr1a in developmental lineages contributing to congenital anomalies in affected individuals were examined via conditional mutagenesis in neural crest cells (face and heart), the second heart field (cardiac outflow tract and right ventricle), and forebrain precursors in mice. Consistent with its ubiquitous role in the essential function of ribosome biogenesis, we observed that loss of Polr1a in any of these lineages causes cell-autonomous apoptosis resulting in embryonic malformations. Altogether, our work greatly expands the phenotype of human POLR1A-related disorders and demonstrates variant-specific effects that provide insights into the underlying pathogenesis of ribosomopathies

NIHR Liver/Renal Biomarker Programme Final Report: Evaluating the benefits for patients and the NHS of new and existing biological fluid biomarkers in liver and renal disease

Protein biomarkers are naturally occurring substances that can be measured, often in fluids such as blood or urine, and which provide information about a patient and their illness. Different diseases have different biomarkers. When people become ill, changes in biomarker levels may occur before any clinical symptoms or signs become apparent. Measuring biomarkers in blood or urine is simple, safe and may help the doctor diagnose which disease the patient has, determine how severe it is, help choose the best treatment and help detect if the disease is getting worse or better. Unfortunately, for many diseases there are not enough biomarkers that are of proven usefulness in patient care today. New developments in research mean that many more are now being discovered but there is no quick and reliable way to decide which of the markers are good enough to be useful clinically. While our research proposal focusses on diseases of the liver and kidney, in the future it can also serve as the "blueprint" for similar work in other diseases. It is aimed at developing a structure and methods to assess the clinical usefulness of biomarkers as quickly and efficiently as possible. The research is divided into three parallel workstreams : 1. Identification of the best research methods for monitoring disease or treatment with biomarkers - the lack of understanding this has hampered this field so far. 2. The creation of a sample "banking" system for collecting and storing patient samples and relevant clinical data from large numbers of patients. This will allow the immediate testing of potential new biomarkers now and in the future. The best biomarkers would then go on to full trials to see if patients and the NHS would benefit from their use. 3. A clinical trial at multiple hospitals in the UK of three new biomarkers for liver damage (together called the "Enhanced liver fibrosis" or "ELF", test). We will find out if ELF can give early warning of dangerous liver damage (cirrhosis) and therefore reduce the risk of major complications. This trial may radically alter the way in which patients with liver disease can be looked after clinically. This research programme will benefit patients and the NHS by ensuring that biomarkers in the future can be evaluated and introduced more rapidly, improving clinical management for each individual patient and leading to better use of NHS resources